Aerosol and topical administration of a formulation containing cyclodextrin, quercetin and zinc, in combination or separately, to mitigate infection by enveloped viruses

ABSTRACT

The invention provides the use of a formulation containing cyclodextrine, quercetin and zinc, at appropriate concentrations to mitigate infections by enveloped viruses like SARS-CoV-2, influenza and HIV/AIDS, when administered via pulmonary and dermal route. While the different forms of cyclodextrin prevent the entry of coated virus into host cells by extracting and sequestering cholesterol molecules at the virus coat and at the host cell plasma membrane, the natural plant-based ionophore quercetin in the formulation, enables cellular entry of zinc, inhibiting viral replication by altering polymerase activity in the host cell. Hence cyclodextrine, and quercetin plus zinc, either in combination or in tandem order of administration, serves both as prophylactic and therapeutic.

RELATED U.S. APPLICATION DATA

Continuation-in-part of provisional application No. 63/235,772 filed onAug. 22, 2021

REFERENCES CITED

US Patent Documents: U.S. Pat. No. 9,034,846 B2 May 19, 2015

US Patent Documents: U.S. Pat. No. 8,440,704 B2 May 14, 2013

FIELD

Medicine

OTHER PUBLICATIONS

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SUBSTANCE

Formulation is adapted for both surface use and introduction to asubject by dispersal, utilizing any known method of measured nasalsprays or inhalers, wet wipes and medicated masks. The formulation isintroduced in form of an aqueous phosphate buffered saline pH 7.5solution, containing 0.01% Benzylkonium chloride as preservative anddifferent concentrations of the active ingredients: 1-5% cyclodextrin;the flavonoid quercetin, a naturally occurring zinc ionophore at aconcentration of 8 ug/ml; and 1 mg/ml zinc chloride. Depending onrequirement, pH 2.5 citrate buffered aqueous will also be used, wherethe low pH serves as a preservative and a solvent for cyclodextrin. Theactive ingredient cyclodextrin (CD): is currently being used as anexcipient in pharmaceutical products including in nasal sprays. The U.S.Food and Drug Administration (FDA) has approved the use of CDs since2001. CDs were first employed in the the food industry in the 1970s, andsince they have been used as food additives for carrying food-relatedlipophiles such as vitamins, aromas and colorants. The firstpharmaceutical patent related to CDs and pharmaceutical applicability,was made in 1953 to serve as a complexing agents. Pharmaceuticalproducts containing CDs comprise nasal spray, oral solutions, soliddosage forms, ocular and dermal formulations, suppositories, andparenteral solutions. Currently, more than 40 pharmaceutical productscontaining CDs are available in the market worldwide, and the vastmajority of them utilize βCD and its derivatives having higher watersolubility such as HPβCD, MβCD, and SBEβCD. Most of the βCD are alsoapproved by the European Medical Agency for all human administrationpathways. The active ingredients zinc and quercetin: Zinc is anessential trace element supporting growth, development and immunehealth. Zinc is also known to protect against viruses by inhibiting RNAbinding, RNA synthesis, viral polyprotein cleavage, viral replication,and viral protease enzyme inactivation. Zinc however needs to enter thehost cell to protect against the virus. Quercetin, a naturally occurringplant-based over the counter zinc ionophore, will enable the cellularentry of zinc to protect host cells against the virus. Furthermore,quercetin has shown therapeutic effects against influenza virus.Additionally, in silico modelling of the interactions between theSARS-CoV-2 Viral Spike Protein and the epithelial cell AngiotensinConverting Enzyme-2 (ACE2) protein, has identified quercetin from adatabase of 8,000 small molecule candidates of known drugs, metabolites,and natural products, as one of the top 5 most potent compounds forbinding to the interface site, and disrupt initiation of viralinfection.

BACKGROUND OF INVENTION

Viruses enter hosts via the epithelium. The cell plasma membrane of skinand lung epithelia is the first line of defence and when breached,serves as the portal for viral entry into hosts. Studies in the past twodecades report the various cell membrane binding (1-9) and entry (10-15)mechanisms utilized by viruses to infect. Irrespective of the differentmechanisms involved in viral entry into host cells, the initiatingcritical process is binding of the virus to the cell plasma membrane.Without binding of virus to the cell plasma membrane, there would be noviral entry in to the host.

A large number of studies have established that binding of viruses tothe cell plasma membrane is subjected to the presence of docking sitesor receptors and their regulation by membrane lipid composition anddistribution such as the establishment of domains called rafts (7). Ourrecent study involving cellular membrane biogenesis, demonstrate thatchanges in composition of membrane cholesterol, impacts both thechemistry and distribution of plasma membrane proteins and lipids,impacting cell function (16). In this study (16) we report that cellsexposed to an increasing concentration of methyl beta cyclodextrin(M-βCD) to deplete cholesterol from the cell plasma membrane demonstrateloss in the uptake of phosphotidyl serine by the cell plasma membrane,while the uptake of phosphatidylethanolamine remain unchanged.Similarly, the loss of cholesterol from the cell plasma membraneresulted in the depletion of membrane fusion proteins such as syntaxinand SNAP25 from the plasma membrane suggesting altered membranefusogenicity. Therefore changes to the chemistry of the epithelial cellplasma membrane via depletion of sterols/cholesterol by cyclodextrins(CDs), could dictate both the binding of the virus at the cell plasmamembrane, and influence both the efficacy and potency of its entry intothe host cell.

In agreement, recent studies (17,18) demonstrate that depletion ofplasma membrane cholesterol in host cells using M-βCD, significantlyreduces entry of the pseudorabies and vaccinia virus into cells.Similarly, studies (19-21) demonstrate that HIV infectivity iscritically dependent on cholesterol. Cholesterol microdomains, calledlipid ‘rafts’, have been suggested in the cellular entry or infection ofHIV, its assembly, and its release from infected cells. Studies furtherreport that plasma membrane cholesterol is also required for a widerange of both bacterial (22,23) and yeast infections (24). Furthermore,high-cholesterol diet impairs pulmonary host defence againstgram-negative bacteria and Mycobacterium tuberculosis (25,26). Takentogether, these results support that CD-mediated depletion of plasmamembrane cholesterol in epithelial cells i.e., skin, nasal passage andlung epithelia in humans, using topological application, aerosol sprayand nebulization, will mitigate both viral entry and secondary bacterialand yeast infections.

CDs are a family of cyclic oligosaccharides constituted of a macrocyclicring of glucose subunits joined by α-1,4 glycosidic bonds (27). CDs areused for improving the water-solubility and bioavailability of a widerange of drugs drugs. The U.S. Food and Drug Administration (FDA) hasapproved the use of cyclodextrins since 2001. Cyclodextrins were firstemployed in the the food industry in the 1970s, and since they have beenused as food additives for carrying food-related lipophiles such asvitamins, aromas and colorants (28). βCD has also been used as acholesterol-reducing agent in food of animal origin such as milk and egg(29,30). The first pharmaceutical patent related to CDs andpharmaceutical applicability as complexing agents is dated 1953 (31).Currently, cyclodextrins are employed in pharmaceutical productsprimarily to increase water solubility of poorly soluble drugformulations and to enhance drug bioavailabilities. Pharmaceuticalproducts containing CDs comprise nasal spray, oral solutions, soliddosage forms, ocular and dermal formulations, suppositories, andparenteral solutions (32). Currently, more than 40 pharmaceuticalproducts containing CDs are available in the market worldwide, and thevast majority of them utilize βCD and its derivatives having higherwater solubility such as HPβCD, MβCD, and SBEβCD (33). Most of the βCDare also approved by the European Medical Agency for all humanadministration pathways (34). CDs are used for example in tablets,aqueous parenteral solutions, nasal sprays and eye drop solutions.Examples of the use of cyclodextrins in medicines on the European marketare R-CD in cetirizine tablets and cisapride suppositories, γ-CD inminoxidil solution, and examples of the use of β-cyclodextrinderivatives are SBE-β-CD in the intravenous antimycotic voriconazole,HP-β-CD in the antifungal itraconazole, intravenous and oral solutions,and RM-β-CD in a nasal spray for hormone replacement therapy by17β-estradiol. In Germany and Japan there are infusion products on themarket, containing alprostadil (prostaglandin E1, PGE1) with α-CD.Cyclodextrins are currently not included in the European CommissionGuideline on excipients in the label and package leaflet of medicinalproducts for human use (35-37). CDs in combination with quercetin andzinc, or used separately in tandem order (CDs followed by Quercetin+Zn),either via pulmonary or dermal route, have never been used as ananti-viral drug until now, as presented here in this application.

Formulation to enhance CD stability and preserve sterility Benzalkoniumchloride widely used as a preservative in nasal sprays and nebulization,has been reported to cause sinonasal mucosal injury (38-40), nasalsquamous metaplasia (39), ciliary dysmotility (41,42), genotoxicity(43,44), and other adverse effects (45-47). Data also suggests the toxiceffects of phenylcarbinol, another commonly used preservative (48-50).Despite this evidence, these preservatives continue to be used at higherconcentrations even in over the counter preparations. Acidification (pH2.5) of nasal, inhalable, and topical ophthalmic preparations have beendemonstrated to maintain sterility without the need for preservatives(51,52). This approach of lowering the pH of the formulated CD andquercetin solutions to be used in wipes and aerosol sprays, willpreclude the use of harmful preservatives at higher concentrations,without compromising sterility of the formulation. Therefore, either lowconcentration of benzalkonium chloride and or low pH formulations willbe prepared for use.

Natural CDs such as αCD, βCD, and γCD are hydrophilic in aqueoussolutions, however they tend to self assemble and form complexes. Toovercome this limitation, soluble βCD derivatives such as2-hydroxypropyl-βCD (HPβCD) and sulfobutylether βCD sodium salt(SBEβCD), are preferred for use in aqueous pharmaceutical formulations(53). Studies report that inorganic acids such as phosphoric and citricacid induce CD solubilization (54,55).

Additional Active Ingredients Zinc and Quercetin

Zinc is an essential trace element supporting growth, development andimmune health, Zinc is also known to protect against viruses: byinhibiting RNA binding, RNA synthesis, viral polyprotein cleavage, viralreplication, and viral protease enzyme inactivation (56). Zinc howeverneeds to enter the host cell to protect against the virus. Quercetin, anaturally occurring plant-based over the counter zinc ionophore, willenable the cellular entry of zinc to protect host cells against thevirus. Furthermore, quercetin has shown therapeutic effects againstinfluenza virus (57-64). Additionally, in silico modelling of theinteractions between the SARS-CoV-2 Viral Spike Protein and theepithelial cell Angiotensin Converting Enzyme-2 (ACE2) protein, hasidentified quercetin from a database of 8,000 small molecule candidatesof known drugs, metabolites, and natural products, as one of the top 5most potent compounds for binding to the interface site, and disruptinitiation of viral infection (65).

Therefore, our CD formulation will utilize FDA approved concentrationsof CDs, quercetin and zinc in buffered solutions to retain both highsolubility and sterility. Mode of administration will be through aerosolspray and nebulization, and topical application on body surface using awaterbased solution adsorbed to paper, cellulose or fabric (66). Thetopical application on body surfaces will including the face and neck,to mitigate envelop virus (such as SARS-CoV-2, influenza and HIV),bacteria and fungus infections.

INVENTION

The invention provides the use of a formulation containingcyclodextrine, quercetin and zinc, at appropriate concentrations tomitigate infections by enveloped viruses like SARS-CoV-2, influenza andHIV/AIDS. While the different forms of cyclodextrin prevent the entry ofcoated virus into host cells by extracting and sequestering cholesterolmolecules at the virus coat and at the host cell plasma membrane, thenatural plant-based ionophore quercetin in the formulation, enablescellular entry of zinc, inhibiting viral replication by alteringpolymerase activity in the host cell.

Using the non-toxic U.S. Food and Drug Administration (FDA)-approvedexcipient cyclodextrin as a drug in phosphate buffered saline solutions,will allow the extraction of cholesterol molecules from enveloped virusmembranes and the host cell membrane, altering their respective lipidand protein composition and distribution, preventing virus entry intohost cells. Using quercetin, a naturally occurring plant-based over thecounter zinc ionophore, will enable the cellular entry of zinc toprotect host cells against the virus by inhibiting RNA binding, RNAsynthesis; viral polyprotein cleavage, viral replication, and viralprotease enzyme inactivation, among others. Administration ofcyclodextrin alone followed by quercetin and zinc administration or thetwo combined, in a waterbased soluble formulation, prevents both viralentry and replication in host cells. Aerosol spray and nebulization ofthe combined cyclodextrin, quercetin and zinc, or their tandemadministration as a aqueous phosphate buffered saline pH 7.5 solutioncontaining 0.01% Benzylkonium chloride as preservative, will be used toprotect the airways including lungs from all coat virus infections.Similarly, topical application of the combined cyclodextrin, quercetinand zinc in aqueous phosphate buffered saline pH 7.5 solution containing0.01% Benzylkonium chloride as preservative, will be used to protectbody surface (skin) from all coated virus infection. Additionally,application to both sides of cellulose masks of the combinedcyclodextrin, quercetin and zinc aqueous phosphate buffered saline pH7.5 solution containing 0.01% Benzylkonium chloride as preservative,will further protect the airways including lungs from all coated virusinfection. In such medicated masks, any airborn droplets containing thevirus will be neutralized on contact with the medicated mask. This isthe first direct use of cyclodextrine, quercetin and zinc as ananti-viral, anti-bactericidal and antifungal drug, either in combinationor used separately in tandem order (CDs followed by quercetin+Zn) viapulmonary or dermal route.

As a proof, recent clinical studies separately using CDs (67) andquercetin (68) on humans, some using randomized, controlled and openlabel clinical trial, show 98%-100% effective in providing protectionfrom SARS-CoV-2 infection as determined using RT-qPCR. These studiesshow that 98% of SARS-CoV-2 infected patients were cleared of the virusin just two weeks of receiving the quercetin treatment.

1. It is claimed that by using the non-toxic U.S. Food and DrugAdministration (FDA)-approved excipient cyclodextrin as a drug inphosphate buffered solutions, will allow the extraction of cholesterolmolecules from enveloped virus membranes and the host cell membrane,altering their respective lipid and protein composition anddistribution, preventing virus entry into host cells.
 2. It is claimedthat by using quercetin, a naturally occurring plant-based over thecounter zinc ionophore, will enable the cellular entry of zinc toprotect host cells against the virus by inhibiting RNA binding, RNAsynthesis, viral polyprotein cleavage, viral replication, and viralprotease enzyme inactivation, among others.
 3. It is claimed that bycombining cyclodextrin, quercetin and zinc in a waterbased solubleformulation, both viral entry and replication will be preventable.
 4. Itis claimed that aerosol spray and nebulization of the combinedcyclodextrin, quercetin and zinc as a aqueous phosphate buffered salinepH 7.5 solution containing 0.01% Benzylkonium chloride as preservative,will be used to protect the airways including lungs from all coatedvirus infection.
 5. It is claimed that the pulmonary administration ofcyclodextrins, serve as prophylactic, preventing entry of SARS-CoV-2into lung epithelial cells, while the ionophore quercetin, enablescellular entry of zinc, inhibiting viral replication, hence istherapeutic.
 6. It is claimed that quercetin and zinc administered viathe pulmonary route, could rapidly enter the blood stream and protectother tissues in the body from SARS-CoV-2.
 7. It is claimed thatlowering the pH of the formulated CD and quercetin-Zn solutions to 2.5,and or low concentrations (0.01%) of Banzylkonium chloride be used inwipes and aerosol sprays, that will preclude the use of harmfulpreservatives at higher concentrations, without compromising sterilityof the formulation.
 8. It is claimed that topical application of thecombined cyclodextrin, quercetin and zinc in aqueous phosphate bufferedsaline pH 7.5 solution containing 0.01% Benzylkonium chloride aspreservative, will be used to protect body surface (skin) from allcoated virus infection.
 9. It is claimed that application to both sidesof cellulose masks of the combined cyclodextrin, quercetin and zincaqueous phosphate buffered saline pH 7.5 solution containing 0.01%Benzylkonium chloride as preservative, will further protect the airwaysincluding lungs from all coated virus infection. In such medicatedmasks, any airborn droplets containing the virus will be neutralized oncontact with the medicated mask.